[Analysis of genetic variant in a patient with juvenile meterochromic leukodystrophy].

OBJECTIVE: To explore the genetic basis for a child with metachromatic leukodystrophy (MLD). METHODS: Clinical data of the patient was collected. Genomic DNA was extracted from peripheral blood samples of the child and his family members. Potential variant was screened by whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing. The pathogenicity the variant was analyzed by multiple sequence alignment of the amino acid sequence and three-dimensional model prediction of its protein product. RESULTS: The child was found to harbor compound heterozygous variants c.257G>A (p.R86Q) and c.467del (p.G156Afs*6) of the ARSA gene, among which the c.467del (p.G156Afs*6) frameshift variation was unreported previously. Multiple sequence alignment showed that the site of the c.257G>A (p.R86Q) missense variant is highly conserved. Three-dimensional structure modeling analysis showed that the partial deletion due to the p.G156Afs*6 variant may cause significant alteration of the structure of ARSA protein. CONCLUSION: The discovery of novel variant in ARSA has enriched the mutational spectrum of MLD and may facilitate the understanding of the genotype-phenotype correlation of MLD.

Mutations in ASH1L confer susceptibility to Tourette syndrome.

Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by repetitive motor movements and vocal tics. The clinical manifestations of TS are complex and often overlap with other neuropsychiatric disorders. TS is highly heritable; however, the underlying genetic basis and molecular and neuronal mechanisms of TS remain largely unknown. We performed whole-exome sequencing of a hundred trios (probands and their parents) with detailed records of their clinical presentations and identified a risk gene, ASH1L, that was both de novo mutated and associated with TS based on a transmission disequilibrium test. As a replication, we performed follow-up targeted sequencing of ASH1L in additional 524 unrelated TS samples and replicated the association (P value = 0.001). The point mutations in ASH1L cause defects in its enzymatic activity. Therefore, we established a transgenic mouse line and performed an array of anatomical, behavioral, and functional assays to investigate ASH1L function. The Ash1l+/- mice manifested tic-like behaviors and compulsive behaviors that could be rescued by the tic-relieving drug haloperidol. We also found that Ash1l disruption leads to hyper-activation and elevated dopamine-releasing events in the dorsal striatum, all of which could explain the neural mechanisms for the behavioral abnormalities in mice. Taken together, our results provide compelling evidence that ASH1L is a TS risk gene.

Association between polymorphisms rs2228001 and rs2228000 in XPC and genetic susceptibility to preeclampsia: a case control study.

BACKGROUND: Xeroderma pigmentosum complementation group C (XPC) is a DNA damage recognition protein that plays an important role in nucleotide excision repair and can reduce oxidative stress, which may be involved in the development of preeclampsia (PE). Therefore, the aim of this study was to explore whether XPC polymorphisms were relevant to the genetic susceptibility to PE in Chinese Han women. METHOD: A total of 1276 healthy pregnant women were included as the control group and 958 pregnant women with PE as the case group. DNA was extracted from peripheral blood samples to perform genotyping of loci rs2228001 and rs2228000 in XPC through real-time quantitative polymerase chain reaction (PCR). The relationship between XPC and susceptibility to PE was evaluated by comparing the genotypic and allelic frequencies between the two groups of pregnant women. RESULTS: Polymorphism of rs2228000 may be associated with PE risk and allele T may play a protective role (genotype, χ2 = 38.961, P < 0.001 and allele χ2 = 21.746 P < 0.001, odds ratio (OR) = 0.885, 95% confidence interval (CI) = 0.840-0.932). No significant difference was found between the two groups in rs2228001,(genotype χ2 = 3.148, P = 0.207 and allele χ2 = 0.59, P = 0.442, OR = 1.017, 95% CI = 0.974-1.062). When the frequencies of genotypes and alleles for early- and late-onset PE, mild PE and severe PE were compared with those of controls, the results were consistent with the large clinical sample. CONCLUSION: Our data suggest that the genetic variant rs2228000 in XPC may be associated with PE risk in Chinese Han women, and that pregnant women with the TT genotype have a reduced risk of PE. Further investigations are needed to confirm these findings in other regions or larger prospective populations.

Novel THRB mutation analysis in congenital hypothyroidism with thyroid dysgenesis.

Thyroid dysgenesis (TD) accounts for most cases of congenital hypothyroidism. Although mutations in thyroid hormone receptor ß (THRB) have been identified in TD, the mutational spectrum of THRB and phenotype-genotype correlations have not been fully elucidated. In this study, we aimed to find mutations of THRB, examine the functions of these mutations, and attempt to elucidate the relationship between THRB and TD. Thus, we screened the exons of THRB in 280 patients with TD and 200 normal subjects in samples collected from China. We performed cell morphology assays, MTT assays, flow cytometric analyses, and a quantitative reverse-transcription polymerase chain reaction in human thyroid follicular epithelial cells (Nthy-ori cell line) to examine the impact of THRB mutations. In two unrelated patients, two novel missense mutations, c.76G>A (p.D26N) and c.107G>A (p.C36Y), were identified in THRB. Functional studies suggested that the C36Y mutant caused changes in morphology, inhibiting cell proliferation and promoting apoptosis in a human thyroid cell line. In addition, we found that messenger RNA expressions of thyroglobulin (TG) and the Na+ /I- symporter (NIS) were decreased in a time-dependent manner in mutant THRB compared with the wild type. To our knowledge, this is the first study to document the prevalence of THRB mutations and the genotype-phenotype spectrum of TD in a Chinese population. We characterized the function of a C36Y mutation, which reduced cell proliferation and increased cell death in thyroid epithelial cells. This study provides further evidence for genetic THRB defects and disease mechanisms in TD.

Association of NLRP3 and IL-4 VNTR polymorphisms and genetic susceptibility to preeclampsia: A case-control study.

INTRODUCTION: Abnormalities in the maternal immune system and insufficient gestational immune tolerance may significantly contribute to the development of preeclampsia (PE). The NLR family pyrin domain containing 3 (NLRP3) functions as a pattern recognition receptor that identifies pathogen-associated molecular patterns. Interleukin-4 (IL-4) is a potent anti-inflammatory cytokine that modulates the immune response. Therefore, this study aims to elucidate the impact of NLRP3 and IL-4 variable number of tandem repeats (VNTR) polymorphisms on susceptibility to PE. MATERIALS AND METHODS: A total of 1,018 patients with PE and 1,007 normal pregnant women were recruited as the case group and the control group, respectively. Peripheral blood DNA was extracted, and NLRP3 and IL-4 VNTR polymorphisms were genotyped using polymerase chain reaction and gel electrophoresis. Genotypes and allele frequencies of pregnant women were assessed in both cohorts. RESULTS: The NLRP3 VNTR 9-7 genotype in the PE group was significantly lower than that in the control group, but 9 and 14 allele frequencies were significantly higher in patients with PE. Individuals with IL-4 VNTR genotypes 1-2 had a lower risk of PE than controls, and the IL-4 VNTR 2 allele frequency was significantly lower in patients with PE. CONCLUSIONS: This study, the first of its kind in the literature, evaluates the impact of NLRP3 VNTR and IL-4 VNTR polymorphisms on PE, revealing a significant correlation with PE susceptibility. This investigation contributes to understanding the pathogenesis of PE and provides a reference point for developing strategies to prevent and treat the disease in the future.

A colorimetric method for H1N1 DNA detection using rolling circle amplification.

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay has been successfully developed as a method for the effective detection of H1N1 DNA. Specific oligonucleotide and reporter primer probes were designed together with a circular template, and the oligonucleotide probes were attached to the surfaces of magnetic beads (MBs) to form functional MB-DNA conjugates as capture probes for the target H1N1 DNA molecules. Together with the addition of DNA targets and reporter primer probes to the MB-DNA conjugates, sandwiched hybrids were formed. The initiation of RCA amplification using the circular template in the presence of phi29 polymerase allowed for the amplification of a large number of repeat sequences of the single-stranded (ss)-DNA product. This RCA product accumulated gold nanoparticles (AuNPs), resulting in a colorimetric change that could be viewed by the naked eye or detected using UV-vis spectroscopy. According to this method, H1N1 DNA could be detected at the 1 pmol L(-1) level. This platform exhibited design convenience, simplicity, and cost-effectiveness, and could be used to provide a new diagnostic assay for H1N1, and other infectious diseases.

DUOX2 and DUOXA2 Variants Confer Susceptibility to Thyroid Dysgenesis and Gland-in-situ With Congenital Hypothyroidism.

Background: Thyroid dysgenesis (TD), which is caused by gland developmental abnormalities, is the most common cause of congenital hypothyroidism (CH). In addition, advances in diagnostic techniques have facilitated the identification of mild CH patients with a gland-in-situ (GIS) with normal thyroid morphology. Therefore, TD and GIS account for the vast majority of CH cases. Methods: Sixteen known genes to be related to CH were sequenced and screened for variations by next-generation sequencing (NGS) in a cohort of 377 CH cases, including 288 TD cases and 89 GIS cases. Results: In our CH cohort, we found that DUOX2 (21.22%) was the most commonly variant pathogenic gene, while DUOXA2 was prominent in TD (18.75%) and DUOX2 was prominent in GIS (34.83%). Both biallelic and triple variants of DUOX2 were found to be most common in children with TD and children with GIS. The most frequent combination was DUOX2 with DUOXA1 among the 61 patients who carried digenic variants. We also found for the first time that biallelic TG, DUOXA2, and DUOXA1 variants participate in the pathogenesis of TD. In addition, the variant p.Y246X in DUOXA2 was the most common variant hotspot, with 58 novel variants identified in our study. Conclusion: We meticulously described the types and characteristics of variants from sixteen known gene in children with TD and GIS in the Chinese population, suggesting that DUOXA2 and DUOX2 variants may confer susceptibility to TD and GIS via polygenic inheritance and multiple factors, which further expands the genotype-phenotype spectrum of CH in China.

Genetic testing of PAX8 mutations associated with thyroid dysgenesis in Chinese congenital hypothyroidism patients.

INTRODUCTION: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH), affecting nearly 1 in 2000-3000 newborns worldwide, as the most common neonatal endocrine disorder. Paired box gene 8 (PAX8), expressed during all stages of thyroid follicular cell, plays a key role in thyroid morphogenesis by a complex regulatory network. In conclusion, the genetic mechanism of PAX8 mutant in TD is still ambiguous; therefore, further research is needed. MATERIAL AND METHODS: Blood samples were collected from 289 TD patients in Shandong Province, China. Genomic DNA was extracted from peripheral blood. All the exons of PAX8 along with their exon-intro boundaries were amplified by PCR and analysed by Sanger sequencing. RESULTS: We identified three novel PAX8 nonsense mutations in three patients by sequence analysis of PAX8: Patient 1 (c.285C>G, p.Tyr95Ter), Patient 2 (c.747T>G, p.Tyr249Ter), and Patient 3 (c.786C>A, p.Tyr262Ter). All the three patients carrying PAX8 variants had obvious clinical phenotypes of thyroid anomaly, such as hypoplasia and athyreosis. CONCLUSION: We conducted the largest worldwide PAX8 mutation screening so far in TD patients. Three presumably pathogenic PAX8 mutations were detected in 289 TD cases for the first time, showing the mutation rate of PAX8 is 1.04% in Chinese TD patients. In addition, our study expands the gene mutation spectrum of TD.

Identification of Two Missense Mutations in DUOX1 (p.R1307Q) and DUOXA1 (p.R56W) That Can Cause Congenital Hypothyroidism Through Impairing H2O2 Generation.

Context: The DUOX/DUOXA systems play a key role in H2O2 generation in thyroid cells, which is required for iodine organification and thyroid hormone synthesis. DUOX2/DUOXA2 defects can cause congenital hypothyroidism (CH), but it is unknown whether DUOX1/DUOXA1 mutations can also cause CH. Objective: We aimed to identify DUOX1/DUOXA1 mutations and explore their role in the development of CH by investigating their functional impacts on H2O2 generation. Patients and Methods: Forty-three children with CH with goiter were enrolled, in whom all exons and flanking intronic regions of DUOX1/DUOXA1 were directly sequenced. We characterized the functional effects of identified mutations on the expression of DUOX1 and DUOXA1 and H2O2 generation. Results: We identified a heterozygous DUOX1 missense mutation (G > A base substitution at nucleotide 3920 in exon 31) that changed a highly conserved arginine to glutamine at residual 1307 (p.R1307Q) in patient 1. A heterozygous-missense mutation (c.166 C>T; p.R56W) was identified in DUOXA1 in patient 2. Functional studies demonstrated that both p.R1307Q mutant or p.R56W mutant decreased the DUOX1 expression at mRNA and protein levels, with a corresponding impairment in H2O2 generation (P < 0.01). The results also showed that intact DUOXA1 was required for full activity of DUOX1 and H2O2 generation. Conclusions: We have identified two heterozygous missense mutations in DUOX1 and DUOXA1 in two patients that can cause CH through disrupting the coordination of DUOX1 and DUOXA1 in the generation of H2O2. This study for the first time demonstrates that the DUOX1/DUOXA1 system, if genetically defective, can cause CH.